Integrating concept of pharmacophore with graph neural networks for chemical property prediction and interpretation.

Recently, graph neural networks (GNNs) have revolutionized the field of chemical property prediction and achieved state-of-the-art results on benchmark data sets. Compared with the traditional descriptor- and fingerprint-based QSAR models, GNNs can learn task related representations, which completely gets rid of the rules defined by experts. However, due to the lack of useful prior knowledge, the prediction performance and interpretability of the GNNs may be affected. In this study, we introduced a new GNN model called RG-MPNN for chemical property prediction that integrated pharmacophore information hierarchically into message-passing neural network (MPNN) architecture, specifically, in the way of pharmacophore-based reduced-graph (RG) pooling. RG-MPNN absorbed not only the information of atoms and bonds from the atom-level message-passing phase, but also the information of pharmacophores from the RG-level message-passing phase. Our experimental results on eleven benchmark and ten kinase data sets showed that our model consistently matched or outperformed other existing GNN models. Furthermore, we demonstrated that applying pharmacophore-based RG pooling to MPNN architecture can generally help GNN models improve the predictive power. The cluster analysis of RG-MPNN representations and the importance analysis of pharmacophore nodes will help chemists gain insights for hit discovery and lead optimization.

Structure-Based Discovery and Structural Basis of a Novel Broad-Spectrum Natural Product against the Main Protease of Coronavirus.

Over the past 20 years, the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2 emerged, causing severe human respiratory diseases throughout the globe. Developing broad-spectrum drugs would be invaluable in responding to new, emerging coronaviruses and to address unmet urgent clinical needs. Main protease (Mpro; also known as 3CLpro) has a major role in the coronavirus life cycle and is one of the most important targets for anti-coronavirus agents. We show that a natural product, noncovalent inhibitor, shikonin, is a pan-main protease inhibitor of SARS-CoV-2, SARS-CoV, MERS-CoV, human coronavirus (HCoV)-HKU1, HCoV-NL63, and HCoV-229E with micromolar half maximal inhibitory concentration (IC50) values. Structures of the main protease of different coronavirus genus, SARS-CoV from the betacoronavirus genus and HCoV-NL63 from the alphacoronavirus genus, were determined by X-ray crystallography and revealed that the inhibitor interacts with key active site residues in a unique mode. The structure of the main protease inhibitor complex presents an opportunity to discover a novel series of broad-spectrum inhibitors. These data provide substantial evidence that shikonin and its derivatives may be effective against most coronaviruses as well as emerging coronaviruses of the future. Given the importance of the main protease for coronavirus therapeutic indication, insights from these studies should accelerate the development and design of safer and more effective antiviral agents. IMPORTANCE The current pandemic has created an urgent need for broad-spectrum inhibitors of SARS-CoV-2. The main protease is relatively conservative compared to the spike protein and, thus, is one of the most promising targets in developing anti-coronavirus agents. We solved the crystal structures of the main protease of SARS-CoV and HCoV-NL63 that bound to shikonin. The structures provide important insights, have broad implications for understanding the structural basis underlying enzyme activity, and can facilitate rational design of broad-spectrum anti-coronavirus ligands as new therapeutic agents.

Protective effect of gastrodin on peripheral neuropathy induced by anti-tumor treatment with vincristine in rat models.

Cancer is a common disease threatening human health, chemotherapy is widely used in clinical treatment of cancer, but chemotherapy-induced peripheral neuropathy (CIPN) has a relevant impact on life quality of cancer patients. Administration of gastrodin can relieve chronic pain to cancer patients with CIPN and attenuated the inflammatory response by reducing the expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). However, its exact molecular mechanisms remain unclear. In this study, we established an animal model of CIPN using Walker-256 breast cancer cell and vincristine. We found that the mechanical and thermal pain threshold of rats was decreased with treatment of vincristine. Using gastrodin could restore the mechanical and thermal threshold without interfering anti-tumor effect of vincristine. Gastrodin relieved CIPN by inhibiting activation of spinal microglia through Fractalkine (CX3CL1) and its receptor CX3CR1, then inhibited P38/mitogen-activated protein kinase (MAPK) signaling pathway and reduced the expression of inflammatory factor TNF-α and interleukin-1ß (IL-1ß). Taking together, our study demonstrated that gastrodin is a potential drug for the treatment of CIPN and likely to improve cancer patient's life quality.

Notch activation enhances microglial CX3CR1/P38 MAPK pathway in rats model of vincristine-induced peripheral neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) has a adverse impact to the living quality of cancer patients. This side effect of CIPN limit the dose of drug used in many chemotherapies, such as vincristine (VCR). The activation of microglia in the spinal dorsal horn is involved in the occurrence and development of neuropathic pain induced by VCR. Recent study has demonstrated that hypoxia induced microglia activation depends on Notch signaling, and it is involved in the release of many inflammatory related factors in microglia. In this work, we aimed to study that the role of Notch signaling pathway in microglia activation on a VCR-induced neuropathy rat model. Our results showed that the mechanical, thermal and cold pain threshold of rats was decreased by treatment of VCR, but N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), a γ-secretase inhibitor, relieved the hyperalgesia. Molecular analysis showed that activation of Notch signaling pathway increased after nerve injury and that DAPT could significantly inhibit the upregulation of Notch signaling pathway, the activation of microglia, and the release of pro-inflammatory cytokines in the spinal. Taking together, Notch signaling pathway could be a potential therapeutic target to alleviate neuropathic pain.

MAVS O-GlcNAcylation Is Essential for Host Antiviral Immunity against Lethal RNA Viruses.

It is known that lethal viruses profoundly manipulate host metabolism, but how the metabolism alternation affects the immediate host antiviral immunity remains elusive. Here, we report that the O-GlcNAcylation of mitochondrial antiviral-signaling protein (MAVS), a key mediator of interferon signaling, is a critical regulation to activate the host innate immunity against RNA viruses. We show that O-GlcNAcylation depletion in myeloid cells renders the host more susceptible to virus infection both in vitro and in vivo. Mechanistically, we demonstrate that MAVS O-GlcNAcylation is required for virus-induced MAVS K63-linked ubiquitination, thereby facilitating IRF3 activation and IFNß production. We further demonstrate that D-glucosamine, a commonly used dietary supplement, effectively protects mice against a range of lethal RNA viruses, including human influenza virus. Our study highlights a critical role of O-GlcNAcylation in regulating host antiviral immunity and validates D-glucosamine as a potential therapeutic for virus infections.

Novel HIV-1 Non-nucleoside Reverse Transcriptase Inhibitor Agents: Optimization of Diarylanilines with High Potency against Wild-Type and Rilpivirine-Resistant E138K Mutant Virus.

Three series (6, 13, and 14) of new diarylaniline (DAAN) analogues were designed, synthesized, and evaluated for anti-HIV potency, especially against the E138K viral strain with a major mutation conferring resistance to the new-generation non-nucleoside reverse transcriptase inhibitor drug rilpivirine (1b). Promising new compounds were then assessed for physicochemical and associated pharmaceutical properties, including aqueous solubility, log P value, and metabolic stability, as well as predicted lipophilic parameters of ligand efficiency, ligand lipophilic efficiency, and ligand efficiency-dependent lipophilicity indices, which are associated with ADME property profiles. Compounds 6a, 14c, and 14d showed high potency against the 1b-resistant E138K mutated viral strain as well as good balance between anti-HIV-1 activity and desirable druglike properties. From the perspective of optimizing future NNRTI compounds as clinical trial candidates, computational modeling results provided valuable information about how the R(1) group might provide greater efficacy against the E138K mutant.

[Synthesis and antisense properties of 2'ß-F- and 2'α-F-2'-deoxy-uridines modified oligonucleotides with 4'-C-(2-methoxyethoxy) substituent].

Chemical modification is critical for the therapeutic applications of antisense oligonucleotides. Novel 4'-C-MOE and 2'-fluoro- modified monomer 2'-F-4'-C-MOE-ara U and its epimeric 2'-F-4'-C-MOE-r U were synthesized from 2'-fluorinated arabinourine (2'-F-ara U) and 2'-fluorouridine(2'-F-r U), respectively. Their phosphoramidites were synthesized and successfully incorporated into oligodeoxynucleotides. The mismatch discrimination ability of these unnatural monomers and their effect on thermal stability were evaluated in the context of ds DNA and DNA-RNA chimeras. The thermal denaturation studies showed that the incorporation of 2'-F-4'-C-MOE-ara U led to enhanced binding affinity to complementary RNA strand and almost equivalent binding ability to complementary DNA, when compared with 2'-F-4'-C-MOE-r U and 2'-F-ara U modified duplexes. Especially a C-H(…)F-C pseudohydrogen bond was supposed to contribute more binding affinity at uridine-purine steps, meanwhile, 2'-F-4'-C-MOE-ara U had almost the same base discriminatory ability as uridine in ds DNA and DNA-RNA chimeras, while 2'-F-4'-C-MOE-r U was found to have only moderate RNA hybridization ability. However, 2'-F-4'-C-MOE-araU at 3'-end of oligonucleotide could not led to more nuclease hydrolytic stability than that with 2'-F-4'-C-MOE-r U modification. These results demonstrated the feasibility of C4'-MOE modification on 2'-F-ANA and the dramatic effects of the 2'-F substituent, which provides a new approach for further chemical modification of antisense drugs.

Optimization of N-aryl-6-methoxy-1,2,3,4-tetrahydroquinolines as tubulin polymerization inhibitors.

Thirteen new N-aryl 1,2,3,4-tetrahydroquinoline compounds (4a-f, 6a-c, and 8a-d) were synthesized and evaluated for antitumor activity and drug-like properties. Compound 4a exhibited high inhibitory potency with low nanomolar GI50 values of 16-20 nM in cellular assays, including excellent activity against the P-glycoprotein overexpressing cell line KBvin. Compound 4a inhibited colchicine binding to tubulin and tubulin assembly with an IC50 value of 0.85 µM, superior to the reference compound CA4 (1.2 µM) in the same assay. In addition, 4a also exhibited highly improved water solubility (75 µg/mL) and a suitable logP value (3.43) at pH 7.4. With a good balance between antitumor potency and drug-like properties, compound 4a could be a new potential drug candidate for further development. Current results on SAR studies and molecular modeling provided more insight about this class of compounds as tubulin polymerization inhibitors targeting the colchicine site.

A nanoparticle-encapsulated non-nucleoside reverse-transcriptase inhibitor with enhanced anti-HIV-1 activity and prolonged circulation time in plasma.

Non-nucleoside reverse-transcriptase inhibitors (NNRTIs), major components of highly active antiretroviral therapy (HAART), are effective in suppressing viral replication and preventing the progress of HIV-1 infection to AIDS. However, rapid blood clearance in vivo could significantly impair the efficiency of the anti-HIV-1 activity and result in multiple daily doses which might lead to poor patient compliance. Here we attempted to employ biodegradable organic nanoparticles (NPs) to encapsulate DAAN15h, a derivative of 4-substituted 1, 5-diarylaniline with potent anti-HIV activities. Nanoparticles encapsulating DAAN15h (NP-DAAN15h) displayed a spherical shape with a size of 97.01 ± 3.64 nm and zeta potential of -19.1 ± 3.78 mV, and they exhibited a sustained controlled release behavior in vitro. The cellular uptake of NPs on TZM-b1 cells, MT-2 cells and M7 cells, possibly through lipid raft-mediated and energydependent active transport processes, was significantly enhanced. NP-DAAN15h, which possessed no significant in vitro cytotoxicity, showed improved antiviral activity against laboratory-adapted and primary HIV-1 isolates with different subtypes and tropisms, including RT-resistant variants. NP-DAAN15h exhibited a significantly prolonged blood circulation time, decreased plasma elimination rate, and enhanced AUC(0-t). NP-DAAN15h, a nanoparticle-encapsulated NNRTI, exhibits enhanced cellular uptake, improved anti-HIV-1 efficacy and prolonged in vivo circulation time, suggesting good potential for further development as a new NNRTI formulation for clinical use.

Physicochemical property-driven optimization of diarylaniline compounds as potent HIV-1 non-nucleoside reverse transcriptase inhibitors.

Using physicochemical property-driven optimization, twelve new diarylaniline compounds (DAANs) (7a-h, 11a-b and 12a-b) were designed and synthesized. Among them, compounds 12a-b not only showed high potency (EC50 0.96-4.92 nM) against both wild-type and drug-resistant viral strains with the lowest fold change (FC 0.91 and 5.13), but also displayed acceptable drug-like properties based on aqueous solubility and lipophilicity (LE>0.3, LLE>5, LELP<10). The correlations between potency and physicochemical properties of these DAAN analogues are also described. Compounds 12a-b merit further development as potent clinical trial candidates against AIDS.

Optimization of the antiviral potency and lipophilicity of halogenated 2,6-diarylpyridinamines as a novel class of HIV-1 NNRTIS.

Nineteen new halogenated diarylpyridinamine (DAPA) analogues modified at the phenoxy C-ring were synthesized and evaluated for anti-HIV activity and certain drug-like properties. Ten compounds showed high anti-HIV activity (EC50 <10 nM). In particular, (E)-6-(2''-bromo-4''-cyanovinyl-6''-methoxy)phenoxy-N(2) -(4'-cyanophenyl)pyridin-2,3-diamine (8 c) displayed low-nanomolar antiviral potency (3-7 nM) against wild-type and drug-resistant viral strains bearing the E138K or K101E mutations, which are associated with resistance to rilvipirine (1 b). Compound 8 c exhibited much lower resistance fold changes (RFC: 1.1-2.1) than 1 b (RFC: 11.8-13.0). Compound 8 c also exhibited better metabolic stability (in vitro half-life) than 1 b in human liver microsomes, possessed low lipophilicity (clog D: 3.29; measured log P: 3.31), and had desirable lipophilic efficiency indices (LE>0.3, LLE>5, LELP<10). With balanced potency and drug-like properties, 8 c merits further development as an anti-HIV drug candidate.

Design and synthesis of diarylamines and diarylethers as cytotoxic antitumor agents.

Based on a shared structural core of diarylamine in several known anticancer drugs as well as a new cytotoxic hit 6-chloro-2-(4-cyanophenyl)amino-3-nitropyridine (7), 30 diarylamines and diarylethers were designed, synthesized, and evaluated for cytotoxic activity against A549, KB, KB-vin, and DU145 human tumor cell lines (HTCL). Four new leads 11e, 12, 13a, and 13b were discovered with GI(50) values ranging from 0.33 to 3.45µM. Preliminary SAR results revealed that a diarylamine or diarylether could serve as an active structural core, meta-chloro and ortho-nitro groups on the A-ring (either pyridine or phenyl ring) were necessary and crucial for cytotoxic activity, and the para-substituents on the other phenyl ring (B-ring) were related to inhibitory selectivity for different tumor cells. In an investigation of potential biological targets of the new leads, high thoughput kinase screening discovered that new leads 11e, 12 and 13b especially inhibit Mer tyrosine kinase, a proto-oncogene associated with munerous tumor types, with IC(50) values of 2.2-3.0µM. Therefore, these findings provide a good starting point to optimize a new class of compounds as potential anticancer agents, particularly targeting Mer tyrosine kinase.

Design, synthesis, and preclinical evaluations of novel 4-substituted 1,5-diarylanilines as potent HIV-1 non-nucleoside reverse transcriptase inhibitor (NNRTI) drug candidates.

Twenty-one new 4-substituted diarylaniline compounds (DAANs) (series 13, 14, and 15) were designed, synthesized, and evaluated against wild-type and drug resistant HIV-1 viral strains. As a result, approximately a dozen new DAANs showed high potency with low nano- to subnanomolar EC(50) values ranging from 0.2 to 10 nM. The three most promising compounds 14e, 14h, and 15h exhibited high potency against wild-type and drug-resistant viral strains with EC(50) values at the subnanomolar level (0.29-0.87 nM) and were comparable to or more potent than the new NNRTI drug riplivirine (2) in the same assays. Druglike physicochemical property assessments revealed that the most active DAANs (EC(50) < 10 nM) have better aqueous solubility (>1-90 µg/mL at pH 7.4 and pH 2) and metabolic stability in vitro than 2, as well as desirable log P values (<5) and polar surface areas (PSA) (<140 Å(2)). These promising results warrant further development of this novel compound class as potential potent anti-AIDS clinical trial candidates.

Optimization of 2,4-diarylanilines as non-nucleoside HIV-1 reverse transcriptase inhibitors.

The current optimization of 2,4-diarylaniline analogs (DAANs) on the central phenyl ring provided a series of new active DAAN derivatives 9a-9e, indicating an accessible modification approach that could improve anti-HIV potency against wild-type and resistant strains, aqueous solubility, and metabolic stability. A new compound 9e not only exhibited extremely high potency against wild-type virus (EC(50) 0.53 nM) and several resistant viral strains (EC(50) 0.36-3.9 nM), but also showed desirable aqueous solubility and metabolic stability, which were comparable or better than those of the anti-HIV-1 drug TMC278 (2). Thus, new compound 9e might be a potential drug candidate for further development of novel next-generation NNRTIs.

[Metabolite profiling of two anti-HIV lead compounds in rat liver microsomes].

The metabolite profiling of DAPA-7012 and DAAN-4442, the lead compounds from two new kinds of non-nucleoside reverse transcriptase inhibitors (NNRTIs), was performed using an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS), with the assistance of a metabolite data processing software. By utilizing the mass defect filter (MDF) technique, the data acquired from the 0 h-incubation and the 2 h-incubation were compared and analyzed with the MetaboLynx software. After incubation, 14 metabolites of DAPA-7012 and 14 metabolites of DAAN-4442 were found in rat liver microsome. The MS2 spectra for some metabolites were obtained using the MS(E) technique to get fragment ions for structural elucidation. The results indicated that both compounds could undergo extensive metabolism in rat liver microsomes. The major phase I reaction was oxidation/hydroxylation. The major phase II reaction was S-glutathione conjugation. The metabolic pathways were similar between the two lead compounds, though they have different backbone structures. Besides, the 4-NO2 of ring B in DAAN-4442 was susceptible to reduction, the benzyl of ring C in DAPA-7012 was tend to be oxidized. The common metabolic soft spots were primary amine of ring B and two methyl groups of ring C. Early SAR results showed that the primary amine and methyl were necessary substituent groups. The stability of these active groups needs to be improved and optimized. The approach of combining metabolites information and structure-activity analysis can provide a reference for further structural optimization.

Design, synthesis, and evaluation of diarylpyridines and diarylanilines as potent non-nucleoside HIV-1 reverse transcriptase inhibitors.

On the basis of the structures and activities of our previously identified non-nucleoside reverse transcriptase inhibitors (NNRTIs), we designed and synthesized two sets of derivatives, diarylpyridines (A) and diarylanilines (B), and tested their anti-HIV-1 activity against infection by HIV-1 NL4-3 and IIIB in TZM-bl and MT-2 cells, respectively. The results showed that most compounds exhibited potent anti-HIV-1 activity with low nanomolar EC50 values, and some of them, such as 13m, 14c, and 14e, displayed high potency with subnanomolar EC50 values, which were more potent than etravirine (TMC125, 1) in the same assays. Notably, these compounds were also highly effective against infection by multi-RTI-resistant strains, suggesting a high potential to further develop these compounds as a novel class of NNRTIs with improved antiviral efficacy and resistance profile.

Diarylaniline derivatives as a distinct class of HIV-1 non-nucleoside reverse transcriptase inhibitors.

By using structure-based drug design and isosteric replacement, diarylaniline and 1,5-diarylbenzene-1,2-diamine derivatives were synthesized and evaluated against wild type HIV-1 and drug-resistant viral strains, resulting in the discovery of diarylaniline derivatives as a distinct class of next-generation HIV-1 non-nucleoside reverse transcriptase inhibitor (NNRTI) agents. The most promising compound 37 showed significant EC(50) values of 0.003-0.032 microM against HIV-1 wild-type strains and of 0.005-0.604 microM against several drug-resistant strains. Current results also revealed important structure-activity relationship (SAR) conclusions for diarylanilines and strongly support our hypothesis that an NH(2) group on the central benzene ring ortho to the aniline moiety is crucial for interaction with K101 of the NNRTI binding site in HIV-1 RT, likely by forming H-bonds with K101. Furthermore, molecular modeling studies with molecular mechanism/general Born surface area (MM/GBSA) technology demonstrated the rationality of our hypothesis.

Discovery of diarylpyridine derivatives as novel non-nucleoside HIV-1 reverse transcriptase inhibitors.

Two series (4 and 5) of diarylpyridine derivatives were designed, synthesized, and evaluated for anti-HIV-1 activity. The most promising compound, 5e, inhibited HIV-1 IIIB, NL4-3, and RTMDR1 with low nanomolar EC50 values and selectivity indexes of >10,000. The results of this study indicate that diarylpyridine can be used as a novel scaffold to derive a new class of potent NNRTIs, active against both wild-type and drug-resistant HIV-1 strains.

[Design, synthesis and biologic evaluation of diarylbenzimidazole derivatives as novel HIV-1 non-nucleoside reverse transcriptase inhibitors].

Twenty seven new diarylbenzimidazole derivatives (A1-A21, B1-B6) were designed, synthesized, and evaluated in MT-2 cell line as potential HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) agents with a new skeleton based on molecular modeling technique and hit 1,2-diarylbenzimidazole A1 (EC50 69.9 miromol x L(-1)). Hence, 1,2-diarylbenzimidazoles A6 and B3, and 1,6-diarylbenzimidazole B6 showed obvious potency against HIV-1 replication in MT-2 cell line with EC50 values of 15.33, 9.81 and 1.37 micromol x L(-1) respectively. All target compounds were synthesized commonly from substituted 2-nitroanilines by 1-3 steps under mild reaction conditions. Current studies provided preliminary SAR, thus indicating that 1,6-diaryl substitution on the benzimidazole ring would be a right direction for further modification. Furthermore, the docking studies demonstrated that B6 could fit well into the HIV-1 NNRTI binding pocket with a similar binding orientation and conformation to that of TMC278, a promising NNRTI candidate inclinical trial III, Therefore, active compound B6 could serve as a new starting point to develop a series of 1,6-diarylbenzimidazole derivatives as HIV-1 NNRTI agents with a novel skeleton.

NMR study on (+/-)-1-[3-(2-methoxyphenoxy)-2-hydroxypropyl]-4-[(2,6-dimethylphenyl)aminocarbonylmethyl]piperazine dihydrochloride salt.

(+/-)-1-[3-(2-Methoxyphenoxy)-2-hydroxypropyl]-4-[(2,6-dimethylphenyl)aminocarbonylmethyl]piperazine dihydrochloride salt was studied spectroscopically. Complete NMR assignments for dihydrochloride salt were made using DEPT, H-H COSY, as well as HMQC and HMBC heteronuclear correlation techniques.